The invention relates to a system and method, using an automated blood cell separator, to prepare a high-quality hemoglobin solution as the raw material for manufacture of hemoglobin-based therapeutic oxygen carriers (xe2x80x9cblood substitutesxe2x80x9d).
The transfusion of stored human blood is an ancient standby of medical practice. However, its efficacy has never been rigorously shown, and the procedure has significant deficiencies. For example, even in the best medical centers, when the need for a transfusion is identified, treatment is delayed by the need to type and cross-match the patient""s blood, then deliver the blood to the patient""s bedside or to the operating or emergency room. In addition, some evidence suggests that blood transfusions may be immunosuppressive and that autologous donor lymphocytes may establish chimeras in the recipient. The risk of transmission of viral diseases by blood transfusion is well-recognized.
In spite of these problems, there is a growing worldwide need for transfusions as medical practice becomes more complex and the population ages. If the rate of transfusion in the United States is extended to the world population of 6 billion, a total annual demand for about 300 million units of red blood cells can be projected. No firm estimate of the number of units actually transfused is available, but the number could be as low as 90 million. Assuming the estimate is accurate, there is a potential shortfall of more than 200 million units per year worldwide. It is not likely that developing countries will be able to support the sophisticated blood banking procedures available in the developed world, nor will most of the developing countries be able to generate sufficient donor blood to meet demands because of the generally poor state of public health.
In an effort to address the potential shortfall in blood supply, therapeutic oxygen carriers, i.e., xe2x80x9cblood substitutesxe2x80x9d, have been under intense development by both commercial and academic laboratories since the mid-1980""s, and even longer in research laboratories. Significant problems have been overcome, including purification of hemoglobin to be used as a raw material, characterization of the solutions, and hemoglobin modification chemistry.
Whether or not a blood substitute succeeds in the marketplace depends on several key factors. First, it must be effective. However, a clear-cut test for efficacy has not been established. One possible test would be whether the product can effectively reduce exposure of patients to allogenic blood. Second, the product must be safe. Safety issues that have arisen to date center around the known property of hemoglobin to be vasoconstrictive. At least part of this property is the very strong binding of nitric oxide (NO) to hemoglobin as a heme ligand and at sulfhydryl sites. Third, a red cell substitute must successfully compete with blood for clinical use. Blood substitute products currently under development have plasma retention times ranging from 12 to 58 hours (half-time). Thus, they will either be used only in temporary situations or in settings where repeated doses can be administered.
Human blood has become extremely safe in the wake of intense scrutiny of the blood bank industry following the discovery that HIV can be transmitted by transfusion of blood products. In order to become a viable product, red cell substitutes must be safe and relatively inexpensive. A cost higher than that of blood will be supported only if there is a clear advantage in safety, efficacy, ease of use, or patient acceptance. Thus, as these blood substitutes near clinical use, the source, cost and supply of raw materials become more important. For products in clinical trials, raw hemoglobin is obtained either from outdated human banked blood, cows, or recombinant (E. coli) sources. Estimates are that one percent or less of stored blood becomes outdated, making only about 120,000 units of blood available for the manufacture of blood substitutes annually. Thus, the competition and cost for this outdated blood is high. Cow blood has the advantage that it can be obtained in large quantities. However, maintaining cows for this purpose requires strict health standards and veterinary care for the animals, frequent testing, and large amounts of land and food to support them. Furthermore, cow blood must be collected using special apparatus designed for that purpose. A recombinant source would be an ideal solution because of the reduced risk of contamination with human pathogens. However, recombinant hemoglobin requires extensive purification to separate the protein from other components of the fermentation, large volumes of water are utilized, and significant problems are encountered in handling the waste products. Such processing requirements result in a blood substitute product made with recombinant hemoglobin costing several times more than the cost of conventionally-banked blood.
The current procedures for preparing hemoglobin solution from human blood involve extensive process of washing pooled red cells with saline, sedimentation or diafiltration, gentle lysis with hypotonic buffers, and rigorous removal of red cell membranes. These procedures require large sterile containers and expensive filters, and take significant lengths of time to complete. Many of the components and solutions used in the process must be housed in cold and/or clean rooms. One reported pilot processing plant with a capacity of 5 liters of stroma-free red blood cell solution per week required four separate but connected rooms covering about 1000 square feet of laboratory space. The cost of production was about $1000/liter. (See Winslow and Chapman, xe2x80x9cPilot-scale preparation of hemoglobin solutionsxe2x80x9d, Meth. Enzymol., 231:3-16, 1994, the disclosure of which is incorporated herein by reference.) In addition to the significant disadvantage of the high cost of production, the large scale and complexity of this pilot set-up required several support personnel and created numerous opportunities for contamination.
In order to make blood substitutes available in the quantities needed to adequately address the projected deficit in worldwide blood supply, the existing methods for processing the raw materials needed to prepare the blood substitutes must be improved. The need remains for a method for preparing stroma-free hemoglobin for use in production of blood substitutes with reduced cost, complexity and risk of contamination of the product.
It is an advantage of the present invention to provide a method for preparation of stroma-free hemoglobin in a self-contained, automated apparatus.
It is another advantage of the present invention to provide a method to prepare a high-quality hemoglobin solution as the raw material for manufacture of blood substitutes using blood obtained at the point of collection, directly from donors, thus eliminating the need for typing and storage of blood, and reducing the risk of contamination.
In an exemplary embodiment, the method employs a commercially-available blood cell separator comprising a computer-controlled centrifuge having a rotor into which a blood processing bag containing donor blood is placed. Once the blood so is collected, the process is performed entirely within the enclosed centrifuge bowl, preferably in situ at the donor collection site. The centrifuge rotor includes one or a more processing chambers for receiving the processing bag(s). In the first step, the blood is centrifuged to separate the plasma from the cellular components. Supernatant, i.e., leukocytes, platelets, and plasma, is removed by using hydraulic fluid force through a flexible diaphragm in the blood processing bag and solenoid-controlled pinch valves, leaving the packed cells. The pinch valves function by pinching the tubing containing the solution, avoiding direct contact to keep the fluid path sterile. A rotating seal allows passage of fluids into and out of the blood processing bag while the centrifuge is rotating. After isolation of the red blood cells from other blood components, the red cells are washed with normal saline or other solution. The wash solution is removed by hydraulic force and transferred through tubing into a supernatant collection container. A photosensor is positioned to monitor the tubing leading to the supernatant collection container to detect the presence of red blood cells. If the red blood cells are detected, a stop or hold function is initiated by the system controller. The red blood cells are then lysed by hypotonic shock and centrifugal force is used to separate the red cell membranes (stroma) from the lysate, which is collected into a sterile container, leaving only the stroma in the centrifuge bowl. The final product can be used as raw material for any of the hemoglobin-based oxygen carriers currently being developed as red cell substitutes. All of these steps are performed within the bowl of the cell separator or saver to maintain sterility.
The ability to process the blood at the donation site provides for more rapid and less costly collection of red blood cells for making blood substitutes. Specifically, this procedure eliminates the need to type and store the units of collected blood, and units collected for this purpose could be pooled to reduce the cost of testing for infectious agents such as HIV or Hepatitis C.